人IκBα基因原核表达质粒的构建及TrxAIκBα融合蛋白的制备

The construction of prokaryotic expression plasmid of human IκBα gene and preparation of TrxA/IκBα fusion protein

目的 利用基因重组技术构建人IκBα基因原核表达质粒 ,制备TrxA IκBα融合蛋白 ,以便进一步研究IκBα的生物学功能和制备相应抗体。方法 以重组质粒pGEM T IκBα为模板 ,利用PCR方法扩增出带有BamHⅠ和HindⅢ酶切位点的人IκBα基因cDNA ,经相应酶切后插入原核表达载体pET 32a(+)。重组表达质粒pET32a(+) IκBα转化大肠杆菌BL2 1(DE3) ,经IPTG诱导表达TrxA IκBα融合蛋白。Westernblot试验鉴定表达蛋白。超声波破菌后采用Ni NTA树脂对TrxA IκBα融合蛋白进行纯化。结果 酶切鉴定证实人IκBα基因cDNA已插入原核表达载体pET 32a(+)。重组表达质粒pET32a(+) IκBα在大肠杆菌BL2 1(DE3)中成功地表达了TrxA IκBα融合蛋白 ,其相对分子质量(Mr)约为 5 6× 10 3 ,表达量约占细菌总蛋白的 2 5 %。Westernblot试验显示TrxA IκBα融合蛋白与兔抗IκBα多克隆抗体呈特异性免疫反应。经Ni NTA树脂纯化后 ,TrxA IκBα融合蛋白的纯度可高达 95 %以上。结论 人IκBα基因原核表达质粒的构建及TrxA IκBα融合蛋白的制备为进一步研究IκBα的生物学功能和制备相应抗体奠定了物质基础。

Objective To construct the prokaryotic expression plasmid of human IκBα gene by recombinant technology and prepare TrxA/IκBα fusion protein for identifing biological function of IκBα and preparing specific antibody against IκBα. Methods Using recombinant plasmid pGEM-T-IκBα as template, the IκBα gene cDNA with BamHⅠand HindⅢ sites was amplified by PCR method and then inserted into the prokaryotic expression vector pET-32a(+) after been digested by corresponding endonucleases. The recombinant expression plasmid pET32a(+)-IκBα was transformed into E.coli. BL21(DE3) and the TrxA/IκBα fusion protein was expressed with induction of IPTG. The expressed protein was identified by Western blot test. The TrxA/IκBα fusion protein was purified with Ni-NTA agarose after sonication. Results Endonucleases digestion confirmed that IκBα gene cDNA has been inserted into the prokaryotic expression vector pET-32a(+). The recombinant expression plasmid pET32a(+)-IκBα successfully expressed TrxA/IκBα fusion protein in E.coli BL21(DE3), and TrxA/IκBα fusion protein showed specific immunological reaction with rabbit anti-IκBα polyclonal antibody. The purity of the TrxA/IκBα fusion protein was over 95% after been purified by Ni-NTA agarose. Conclusion The construction of the prokaryotic expression plasmid of IκBα gene and the preparation of TrxA/IκBα fusion protein established a solid basis for further studying the biological function of IκBα and preparing the specific antibody against IκBα.